To produce biomass fuels including a sugar as a fermentation feedstock, a bioethanol, etc. from a cellulosic biomass such as a wood, rice straw, rice husk or weed, it is required to degrade the main structural component of a plant cell wall, cellulose. To degrade cellulose, an acid saccharification method such as a concentrated sulfuric acid saccharification method or a dilute sulfuric acid saccharification method, an enzymatic saccharification method, etc. may be employed. Because of recent development in biotechnology, research and development of an enzymatic saccharification method are actively carried out.
In the enzymatic saccharification of cellulose, enzymes collectively known as cellulases are utilized. Firstly, an endoglucanase (EG), which has an activity to cleave cellulose chains at random, degrades an amorphous region of cellulose to expose terminal glucose residues. The exposed glucose residues are degraded by a cellobiohydrolase (CBH) to release cellobiose. Thereafter, the released cellobiose is degraded by β-glucosidase (BGL) to release glucose.
For the saccharification of cellulose, filamentous fungi of the genus Aspergillus and the genus Trichoderma are widely used, since they can produce various cellulases and hemicellulases which are required for degrading and saccharifying a crystalline cellulose, and they can secrete a large amount of such enzymes to their extracellular environment.
Further, it has been tried to express such cellulases of filamentous fungi in a heterologous microorganism. Non-patent document 1 discloses that a budding yeast Saccharomyces cerevisiae was transformed with a gene encoding β-glucosidase 1 (BGL 1) of Aspergillus aculeatus to obtain a transformant, and the obtained transformant expressed such an enzyme.
However, in the enzymatic saccharification method, as the enzymatic hydrolysis of cellulose proceeds, glucose accumulates in the reaction system and the accumulated glucose inhibits β-glucosidase, whereby accumulation of cellobiose proceeds. Further, there is a problem such that the complete degradation of cellulose may not be achieved since the accumulated cellobiose inhibits endoglucanase and cellobiohydrolase. Accordingly, development of a highly functional β-glucosidase has been desired.
On the other hand, the genetic analysis of a yeast of the genus Schizosaccharomyces is more advanced than that of a filamentous fungus of the genus Aspergillus or the genus Trichoderma, and the yeast has a lot of advantages like availability of various useful mutants and gene transfer vectors, and its suitability for industrial large-scale production of a protein. However, the yeast of the genus Schizosaccharomyces does not have endogenous β-glucosidase gene, whereby it cannot utilize cellobiose. The present inventors transformed a yeast of the genus Schizosaccharomyces with a gene encoding β-glucosidase thereby to express the enzyme from the obtained transformant (Patent Document 1).
Further, in Non-Patent Document 1, there is no description about an inhibitory effect of glucose to β-glucosidase produced by a budding yeast.
On the other hand, for recovering a β-glucosidase secreted from the transformant, a step of separating a culture broth and cells is required. Although steps of centrifugation, continuous centrifugation, membrane separation, etc. may be mentioned as the separation step, all of them are complicated steps and require a tremendous amount of labor and time.
In addition, it is easily expected that the complexity of the separation step increases as the scale of β-glucosidase production expands.
On the other hand, in the case of using a yeast which exhibits non-sexual flocculation (a property of aggregating non-sexually), aggregated yeast cells are easily separated from a culture broth after cultivation, whereby it is preferred to use a yeast which exhibits non-sexual flocculation as a host cell in the β-glucosidase production.
As the yeast which exhibits non-sexual flocculation, FLO mutants are known for budding yeast Saccharomyces cerevisiae. Further, mutants which exhibit non-sexual flocculation have been reported (e.g. Patent Document 2) for fission yeast Schizosaccharomyces pombe (hereinafter also referred to as S. pombe).
Further, a yeast of the genus Schizosaccharomyces such as S. pombe is phylogenetically quite different from budding yeast Saccharomyces cerevisiae. It is significantly different from other yeasts in its chromosome structure and various mechanisms including genome replication mechanism, RNA splicing mechanism, transcriptional machinery and post-translational modification, and some of them are known to be similar to those of animal cells. Therefore, it has been widely used as a model eukaryote (Non-Patent Document 2).
Because of its various characteristics, S. pombe is considered as a unicellular eukaryote closer to higher animal cells and is a very useful yeast as a host for expression of foreign genes, especially genes derived from higher animals. In particular, it is known to be suitable for expression of genes derived from animals such as human (Patent Documents 3 to 9).
For expressing a protein derived from a foreign structural gene by using S. pombe hosts, usually, a promoter which promotes transcription of the foreign structural gene encoding the protein is required. As the promoter, endogenous promoters for S. pombe genes and promoters from other organisms or viruses are known.
As promoters which have been utilized for the protein expression in S. pombe hosts, promoters endogenous to S. pombe including an alcohol dehydrogenase (adh1) gene promoter, a nmt1 gene promoter involved in thiamine metabolism, a fructose-1, 6-bis phosphatase (fbp1) gene promoter involved in glucose metabolism, an invertase (inv1) gene promoter involved in catabolite repression (Patent Document 7 or 10), a heat shock protein gene promoter (Patent Document 11) may, for example, be mentioned. Further, a promoter of a virus such as hCMV, SV40, or CaMV (constructive expression) is also known (Patent Document 4, 6 or 12).